Topical microbicides are well recognized to be an important part of HIV prevention. However, to be used as a practical regimen, microbicides must meet certain requirements including superior functionality, stability without refrigeration and use acceptability. Protein HIV entry inhibitors are highly promising microbicide candidates with picomolar to nanomolar effectiveness and excellent pre-clinical properties. However, such inhibitors often require refrigeration due to a lack of stability at prolonged high temperatures. W propose a novel strategy to address this challenge using recent advances in the use of an FDA approved biomaterial, silk protein, to stabilize HIV inhibitors without refrigeration. The goal of this project is to study topical HIV microbicides in silk- based film forms that will provide both stability and sustained release of the inhibitors. We will build on our strong preliminary findings that include the stabilization of therapeutics in silk materials, the development of proteins as potent and highly specific viral inhibitors, and the formulation of these proteins with silk. Recen data demonstrate progress in the stabilization of these inhibitors and retention of biological function. The objective is to determine the microbicidal effectiveness of three potent HIV entry inhibitors, 5P12-RANTES, 5P12-RANTES-linker-C37, and Griffithsin, in combination with silk films, to provide stabilization without refrigeration in a film material format for direct use by patients. The hypothesis is that these inhibitors can be formulated into silk materials and retain potency for months without refrigeration and retain excellent pre- clinical properties with the added value of time-release. The goals of the program will be achieved through three specific aims: Specific Aim 1: Formulate HIV inhibitors into silk materials. Specific Aim 2: Determine the effectiveness of silk-formulated inhibitors in primary cell cultures in vitro and mucosal tissue explants (colorectal and cervical). Specific Aim 3: Determine the safety profile of a single silk-formulated inhibitor in vivo using the non-human primate model. Our 2 year go/no go decision point will be based on the retention of 75% efficacy of the HIV inhibitors for at least three months at 50oC, with a formulation capable of sustained release for at least one week. After this point the silk embedded inhibitors will be tested in ex vivo biological assays in colorectal and cervical explant studies. These studies will be followed by continued improvements in stability, as well as sustained release to one month, additional materials format optimization, and in vivo studies to assess safety profile of a silk-formulated inhibitor in the non-human primate model. The proposed studies are highly relevant to the development of effective strategies for the stabilization and delivery of HIV inhibitors for prevention of the viral infection. In summary, the proposed study will capitalize on our collective strengths in silk formulations for drug delivery, producing and testing HIV inhibitors in in vitro cell lines and tissue explants, and the use of the non-human primate model to develop stable HIV inhibitor formulations for prevention of HIV mucosal transmission.